This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The primary focus of this proposal is the bacterial outer membrane protease IcsP (SopA), which is encoded by the virulence plasmid of the enteric pathogen Shigella flexneri. The involvement of IcsP in the pathogenicity of Shigella is poorly understood and warrants further investigation. In specific aim 1, the regulation of icsP will be studied. A virulence gene activator, VirB, up-regulates the icsP promoter. A putative VirB-binding site has been identified within this DNA. This binding-site will be mutated by site-directed mutagenesis and the activity of the icsP promoter will be measured using a lacZ reporter. In vitro experiments will determine whether VirB binds to this site and characterize the mechanism of VirB-dependent regulation of the icsP promoter. Transcription of icsP and IcsP protein levels will be measured under a variety of physiological conditions encountered by Shigella in humans. These studies will provide further insight into how IcsP is regulated and may elucidate how IcsP functions in the pathogenicity of Shigella. In specific aim 2, the possibility that IcsP plays additional roles in Shigella pathogenesis will be explored. The only known substrate of IcsP is another outer membrane protein IcsA. Preliminary data suggest IcsP proteolytically processes at least one other Shigella outer membrane protein. Using 2-D gel electrophoresis and proteomic approaches, this and other outer membrane proteins cleaved by IcsP will be identified. We expect that these studies will improve our understanding of the roles that IcsP plays during bacterial cell growth and differentiation in the host environment. Since proteases expressed by eukaryotes and prokaryotes differ considerably from one another, prokaryotic proteases offer an exciting possibility as targets for drug therapy. An enhanced understanding of IcsP and its role in Shigella growth and differentiation in host environments may allow us to assess whether drug-targeting of this family of proteases is feasible.